Cellular dissection of malaria parasite invasion of human erythrocytes using viable Plasmodium knowlesi merozoites

Abstract

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.

Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention

“…Human erythrocytes were supplied by the Australian Red Cross. P. falciparum (clone 3D7) and P. knowlesi (YH1) were grown in human erythrocytes as previously described [61][62][63][64] . P. falciparum synchronization (4 h window) was achieved using 5% w/v Sorbitol and 15 µl/ml culture of heparin 65 .…”

“…Purification of P. falciparum trophozoite and schizont-infected cells was achieved using a super-MACS column 66 . P. knowlesi cultures for in vitro assays were partially synchronized (∼12 h age range) using heparin 62 .…”

Apicomplexan F‐actin is required for efficient nuclear entry during host cell invasion

“…An Elyra S1 microscope with super-resolution structured illumination microscopy (SR-SIM) (Zeiss) was used for super-resolution dissection of RON4 staining on the merozoite surface. Plasmodium knowlesi A1-H.1 parasites were cultured as described previously [47] in human O+ RBCs in RPMI-HEPES media supplemented with 2.3 g/l sodium bicarbonate, 2 g/l dextrose, 0.05 g/l hypoxanthine, 0.025 g/l gentamicin, 0.292 g/l L-glutamine, 5 g/l Albumax II (Gibco) and 10% (v/v) equine serum (Gibco). Parasites were cultured at 37°C with a gas mixture of 90% N 2 , 5% O 2 and 5% CO 2 .…”

Signalome-wide assessment of erythrocyte response toPlasmodiumreveals novel targets for host-directed antimalarial intervention

“…Purification of P. falciparum trophozoite and schizont-infected cells was achieved using a super-MACS column 71 . P. knowlesi cultures for in vitro assays were partially synchronised (~12-hr age range) using heparin 67 .…”